响应面分析法优化酶解制备酵母肽的工艺研究

为研究酶解处理对啤酒酵母肽得率的影响,文中首先研究了碱性蛋白酶和胰蛋白酶对多肽得率的影响,然后采用响应曲面试验对工艺进行优化。结果表明,胰蛋白酶更适合作为酶解制备酵母肽的工具酶,酶解制备酵母肽的最佳工艺条件是:酶解时间为12h时,温度60℃,pH7.5,底物浓度15.0%,酶添加量0.10%。在此参数条件下做验证实验,检测多肽含量为20.4%。在该工艺条件下,经喷雾干燥,粗粉得率为48%,经测定,其中总氮含量为52.8%,多肽含量为42.6%。     

动物蛋白水解酶制备羽毛多肽的工艺优化

通过正交试验并结合中试试验,利用动物蛋白水解酶,研究不同工艺条件下制备的羽毛多肽粉对肽含量、粗蛋白含量和胃蛋白酶消化率等指标的影响,通过对结果进行极差和方差分析,获得制备羽毛多肽粉的最优条件参数。试验表明,制备羽毛多肽粉的最优条件为:底物浓度8%、pH8.5、酶添加量1.5%酶解时间12h,在此条件下获得羽毛多肽粉肽含量66.89%,粗蛋白含量84.99%,胃蛋白酶消化率达到92.95%。     

富血红素多肽研究进展

动物血液中的血红蛋白占血液总蛋白质含量的60%～70%。利用酶解技术可将血红蛋白降解为具有多种功能活性的生物活性肽,同时释放出富血红素多肽。本文阐述了血红蛋白的分子结构、富血红素多肽的酶法制备及其作用,为利用猪血制备富血红素多肽产品提供一定的参考。     

家蝇蛹的营养成分分析及抗氧化肽制备工艺研究

研究着力于分析家蝇蛹的成分,探索比较家蝇蛹抗氧化肽的制备方法,考察酶解法制备抗氧化肽的工艺条件。实验用水提醇沉法(WAM)、热变性法(TDM)和酶解法(EHM)制备得到家蝇蛹抗氧化肽,检测了三种方法制备产物的得率、应用OPA法测得多肽的含量、聚丙烯酰胺凝胶电泳—银染法表征其分子量分布、比较抗氧化肽在五种体外抗氧化体系(ABTS自由基清除、DPPH自由基清除、羟基自由基清除、FRAP法测还原、亚铁离子螯合)中的抗氧化能力;最后通过单因素试验优化酶解法制备工艺。实验结果显示:水提醇沉法、热变性法和酶解法制备抗氧化肽的得率分别是(18.35±0.43)%、(25.11±0.49)%、(70.80±0.36)%;OPA法检测的多肽含量分别为(53.87±2.74)%、(43.33±1.07)%、(66.50±9.02)%;热变性法制备的抗氧化肽的自由基清除能力和还原能力均较强,酶解法制备抗氧化肽的亚铁离子螯合能力最优,而水提醇沉法的各项指标值均较低。单因素实验结果显示,最佳酶解条件为温度60℃、酶解时间30 min、加酶量6 000 U/g。以上结果提示,家蝇蛹营养成分丰富,酶解法和热变性法均能有效制得强抗氧化活性物质,具有开发成饲料或饲料添加剂的潜力。     

绵羊乳酪蛋白血管紧张素转换酶抑制肽的制备与鉴定

以绵羊乳酪蛋白为原料,采用中性蛋白酶水解制备生物活性肽,研究酶解时间对酶解产物水解度和血管紧张素转换酶（angiotensin converting enzyme,ACE）抑制率的影响,利用液相色谱-质谱联用技术对水解产物进行分析和鉴定,筛选具有潜在ACE抑制作用的生物活性肽。结果表明：酶解时间达到300 min时,水解度最高值达9.65%,ACE抑制率为84.55%;当ACE抑制率达到50%时,所需肽段YYQQRP浓度为5～10 μmol/L;利用超高效液相色谱飞行时间质谱仪对酶解后的多肽进行序列分析,     

桑叶蛋白血管紧张素转换酶抑制肽的酶解制备

本研究旨在探讨桑叶蛋白血管紧张素转换酶(ACE)抑制肽的酶解制备方法,从木瓜蛋白酶、中性蛋白酶、胃蛋白酶、芽孢杆菌蛋白酶、碱性蛋白酶、胰蛋白酶6种蛋白酶中筛选出最佳蛋白酶,并运用单因素逐级优化法对酶解反应的底物浓度、加酶量、温度、pH、酶解时间进行参数优化.选取这6种常用蛋白酶,利用酶解法制备桑叶蛋白多肽,以ACE抑制率为主要指标,水解度为辅助指标,研究桑叶多肽对ACE抑制活性的影响.结果 表明,酶解效果最佳的酶为芽孢杆菌蛋白酶,最佳酶解参数为底物质量浓度20 g/L、加酶量7.5％、温度60℃、pH7.0和酶解时间50 min,此时酶解产物的ACE抑制率为81.51％,水解度为15.86％.     

羽毛粉酶解法制备羽毛肽的工艺研究

试验采用酶解方法处理羽毛粉,制备利于动物机体消化吸收的羽毛肽。羽毛粉经过高温和强碱预处理后,采用不同种类蛋白酶制剂酶解羽毛粉,并优化酶解条件,测定酶解物中可溶性蛋白质含量考察酶解效率;确定最佳制备条件为预处理温度90℃、处理时间150 min及溶解剂质量分数0.7%,得到最佳酶制剂为角蛋白酶,蛋白酶添加量0.8‰、酶解p H 8.5及酶解时间8 h,酶解后低聚肽含量超过70.0%。用高效液相色谱测定酶解后羽毛低聚肽分子质量的分布,约70.0%羽毛肽分子质量≤2 000 D。     

鸡胚酶解多肽制备及其在HDF-α细胞培养中的初步应用

目的:研究制备的鸡胚酶解多肽原液对人真皮成纤维细胞HDF-α的增殖、抗紫外及抗氧作用。方法:将鸡胚去壳、匀浆,破碎细胞;经胰酶酶解后,离心收集上清液,超滤截留分子量小于50 kDa的组分,除菌过滤后为多肽原液;原液进行理化及活性检测,用MTT比色法测定酶解多肽促进上皮细胞增殖,活性氧ROS检测法测定酶解多肽保护细胞抗紫外及抗氧化作用。结果:原液经shotgun分析为来自70种不同蛋白的300余种肽段,含量16 mg/mL,收率91.1%,pH 6.82±0.05,渗透压286 mmol/L,微生物限度检查及细菌内毒素检查合格;鸡胚酶解多肽促HDF-α细胞增殖率为20%～40%,抗紫外效果具有量-效关系,多肽浓度为250 μg/mL时抗紫外保护率约100%,100 μg/mL多肽抗自由基效率高达77%。结论:提取的鸡胚的多肽具有良好的促进HDF-α细胞增殖、抗紫外及抗氧化的细胞保护和促进损伤修复的作用,为鸡胚酶解多肽在HDF-α细胞培养中的作用提供了理论依据。     

碱性蛋白酶酶解藜麦芽制备多肽工艺的研究

为寻找开发藜麦蛋白，提高藜麦应用价值的最佳条件，本研究以白藜麦芽为原料，采用正交试验法优化碱性蛋白酶酶解藜麦芽制备多肽的最佳工艺条件。以碱性蛋白酶添加量、酶解pH、酶解时间、酶解温度和底物浓度这5个因素进行单因素试验，再通过正交试验分析，获得制备多肽的最佳酶解条件为：加酶量2000 U/g，酶解pH 10.0，酶解温度50 ℃，酶解时间3 h，底物浓度9％，在此条件下进行的验证试验，多肽含量达（0.4105±0.0021）mg/mL，表明该工艺稳定、可行，具有较高的应用价值。 [关键词] 藜麦|酶解|多肽|单因素试验|正交设计     

双酶法制备饲料级小麦低聚肽的工艺研究

试验旨在研究使用双酶法制备饲料级小麦低聚肽。试验对比了食品级与饲料级蛋白酶水解谷朊粉的多肽含量，筛选出最佳的复合酶种类；在单因素试验基础上，利用正交试验优化酶解条件并测定可溶性小肽含量。结果显示，酶解工艺条件为谷朊粉添加量12%、pH值7.0、中性蛋白酶与碱性蛋白酶各添加3%、水解5 h。在此条件下，可溶性小肽含量84.34%。使用高效液相色谱测定其重均分子量为764 Da，分子量小于1 000 Da的小麦低聚肽占比87.19%。研究表明，使用饲料级蛋白酶酶解谷朊粉，小麦低聚肽含量显著提高，并降低成本，具有作为功能性饲料添加剂应用的潜力。     

牛初乳酶解物对离体犊牛小肠上皮细胞增殖和吸收功能的影响

初乳经胃蛋白酶水解处理90分钟后所产生的水解物对离体犊牛小肠上皮细胞有明显的促增殖作用(P<0.01) ,而初乳经胰蛋白酶处理则不表现促增殖活性。初乳酶解物对促进细胞吸收葡萄糖的作用随着酶解处理时间的延长而逐渐增强 ,初乳经胰蛋白酶水解处理90分钟或经胃蛋白酶水解处理150分钟时的产物对细胞吸收葡萄糖的促进作用分别达到最强(P<0.01)。试验结果表明 ,初乳酶解物即小分子蛋白质(肽)具有刺激离体小肠上皮细胞增殖和功能发育的活性。     

新疆双峰驼乳酪蛋白血管紧张素转化酶抑制肽的酶法制备和抑制特性

对新疆双峰驼乳酪蛋白分别进行胃蛋白酶和胰蛋白酶的单酶和双酶联合水解,用反相高效液相色谱外标法,对产生的马尿酸含量进行检测,测定水解产物血管紧张素转化酶（angiotensin converting enzyme,ACE）抑制活性。通过米式方程对比水解产物与卡托普利之间的竞争关系,并用50、10、3 kDa的超滤膜对水解液进行超滤,测定截留液ACE抑制活性。结果表明：驼乳酪蛋白单酶水解12 h过程中,胰蛋白酶水解4 h,水解度达到3.7%,胃蛋白酶水解4 h,水解度达到16.58%,胰蛋白酶水解2 h,水解     

海带渣添加比例及其酶解产物对凡纳滨对虾生长、消化和非特异性免疫力的影响

为探究海带渣添加比例及其酶解产物对凡纳滨对虾生长、消化和非特异性免疫力的影响,本试验进行了如下2个试验:海带渣适宜添加比例试验和海带渣酶解产物应用效果试验。海带渣适宜添加比例试验(试验1):采用单因素试验设计,配制海带渣添加比例分别为0(对照组)、0.5%、1.0%、2.0%、3.0%、5.0%的6种饲料(分别记为D1~D6组),投喂初始体重为(1.00±0.10)g的凡纳滨对虾49 d后,对对虾的生长性能指标以及胃消化酶、血清非特异性免疫酶的活性进行测定。每组3个重复,每个重复35尾对虾。海带渣酶解产物应用效果试验(试验2):在试验1得出海带渣适宜添加比例为3.0%的基础上,以试验1中添加3.0%海带渣饲料作为海带渣对照组(A组),再分别用β-葡聚糖酶酶解海带渣(B组)和蛋白酶酶解海带渣(C组)等量替代A组饲料中的海带渣,投喂初始体重为(1.00±0.1)g的凡纳滨对虾56 d,试验结束后测定对虾的生长性能指标以及胃消化酶、血清非特异性免疫酶的活性。每组3个重复,每个重复35尾对虾。试验1结果显示:D4、D5组对虾的特定生长率、增重率显著高于对照组(P0.05),D5组对虾的饲料系数显著低于对照组(P0.05);随着海带渣添加比例的增加,胃中胃蛋白酶及血清中酚氧化酶、超氧化物歧化酶的活性呈现先升高后下降的趋势,上述3种酶的活性均以D5组最高,且D5组血清中酚氧化酶、超氧化物歧化酶的活性显著高于对照组(P0.05)。试验2结果显示:与未经酶解的海带渣相比,海带渣经β-葡聚糖酶酶解后可显著提高对虾的增重率和特定生长率(P0.05),并能显著降低饲料系数(P0.05),海带渣经蛋白酶酶解后可显著提高对虾的成活率(P0.05);B和C组对虾的胃中纤维素酶活性较A组显著降低(P0.05);B和C组血清中酸性磷酸酶、酚氧化物酶活性以及C组血清中超氧化物歧化酶活性显著高于A组(P0.05)。由此得出,凡纳滨对虾饲料中海带渣的适宜添加比例为3.0%。海带渣经β-葡聚糖酶酶解后可促进凡纳滨对虾生长,提高其非特异性免疫力;而海带渣经蛋白酶酶解后可提高凡纳滨的非特异性免疫力,对其生长无促进作用。     

Studies on structural and immunogenic polypeptides of hydropericardium syndrome virus by SDS-PAGE and western blotting

The polypeptide pattern of a local isolate of a virus causing hydropericardium syndrome was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A total of 12 polypeptides ranging in molecular weight between 13.8 and 110.0 kDa were observed. Western blot analysis of structural polypeptides revealed seven immunogenic polypeptides ranging in molecular weight between 15.8 and 110.0 kDa.     

Protein profiles of dense-centered forms of five chlamydial strains of animal origin.

Purified dense-centered form of 1 bovine strain (LW613) and 3 ovine strains (B577, 034-EYE, and 047-EYE) of Chlamydia psittaci and 1 murine strain of Chlamydia trachomatis (MoPn) were dissociated in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol. The number of polypeptides detected in the 5 strains varied between 17 and 20, with a molecular weight range of 29,000 to 120,000. Two polypeptides predominated and comprised approximately a third of the total protein in each of the 5 strains. The average molecular weights of the 2 polypeptides were 89,000 and 85,250 for 4 of the strains, and the polypeptide molecular weights were 100,000 and 98,000 for the ovine abortion strain (B577). Molecular weights and proportional composition of the polypeptides permitted differentiation of the chlamydial strains. The 3 strains from naturally occurring conjunctivitis or polyarthritis (LW613, 034-EYE, and 047-EYE) had similar polypeptide profile in the 75,000 to 100,000 molecular weight range. The polypeptides of the ovine abortion strain (B577) differed significantly from these 3 strains. Eight of the polypeptides of this strain had a molecular weight of 100,000 or greater, and 3 of the predominant polypeptides were in excess of 100,000. In contrast, some of the polypeptides of the murine strain had lower molecular weights than the 4 other strains. Three predominant polypeptides had molecular weights below 50,000.     

Structural proteins of two different plaque-size phenotypes of fowlpox virus

Structural polypeptides of two plaque-purified variant isolates of fowlpox virus differing in plaque morphology and size were examined by Coomassie blue-staining and immunoblot analysis of purified virions. A total of 30 structural polypeptides were observed, ranging in molecular weight from 14,100 to 122,600. A late polypeptide of 36,400 molecular weight was quite prominent in the small-plaque clone but absent in the large-plaque clone. Two other polypeptides, of 33,700 and 34,800 molecular weight, were present in virions from large-plaque virus and cell lysates of both clones but were absent in the small-plaque virions. These differences were observed whether the viruses were grown in chorioallantoic membrane or in chicken embryo fibroblast cultures. No difference was observed between the growth curves of the two virus clones. Differences observed in the polypeptides of the two viruses may be due to changes in the less conserved regions of viral DNA and may be used for differentiation of virus isolates.     

广灵驴分子系谱的建立及群体遗传结构分析

旨在对广灵县优种驴场保种群体进行调查的基础上构建分子系谱,并对其种群的遗传结构进行分析。本研究采集保种群成年、体况良好的广灵驴（体重350~400 kg）颈静脉血10 mL（n=107）,其中公驴13份,母驴94份,抗凝处理后提取全血DNA。采用12个微卫星标记进行荧光PCR扩增后,用ABI3730测序仪进行分型。分型结果采用Cervus 2.0和Pedigraph 2.4软件构建分子系谱,同时采用STRUCTURE2.3和Fstat软件计算群体遗传参数,采用R语言的hclust函数绘制7头公驴及其后代的系统发生NJ树（邻接树）。结果,对107头种驴进行了分子系谱构建,找到了30头子代的父亲和7头子代的母亲,系谱可靠性>90%;微卫星标记的平均观测杂合度（H_Obs）和多态信息含量（PIC）分别为0.676 5和0.593 9,标记遗传多样性较高;NJ树对7个公驴家系进行了聚类;群体分化系数（F_ST）为0.184,群体平均近交系数（F_IT）为0.033,群体内近交系数（F_IS）为-0.238,且群体处于哈代-温伯格平衡状态,存在很弱的近交。本研究建立了广灵驴保种群可靠性较高的分子系谱并对其遗传结构进行了分析,证明该群体遗传多态性较高,群体近交系数较低,处于较好的保种状态,具有较大的品种资源开发潜力。     

Control panels of meat juice samples for a Salmonella enzyme-linked immunosorbent assay.

In the Danish pig production system, an indirect enzyme-linked immunosorbent assay (ELISA) for detection of antibodies in meat juice is used for Salmonella surveillance. Quality control (QC) of this ELISA was previously based on repeated testing of control serum samples. The purpose of the study reported here was to collect, characterize, and implement a panel of meat juice pools for supplemental internal QC. Muscle samples for extraction of meat juice were collected from slaughter pigs of 5 herds infected with Salmonella spp. and from 4 herds without Salmonella infection. A QC panel with 39 pools of meat juice, yielding ELISA optical density (OD) values covering the full range of expected OD values, was prepared and tested repeatedly to determine mean and SD OD values. Each pool was tested twice on each microtitration plate, and the results were used to determine limits for validity of future tests. This QC panel was included as an internal QC to be tested every month. Besides the QC panel, 2 panels containing 100 samples of meat juice with OD above the positive cut-off value and 100 samples with OD below that value were prepared for quarterly control of the diagnostic sensitivity (DSe) and the diagnostic specificity (DSp) of the ELISA. The inclusion of these panels in the QC system will provide information about drifts in DSe and DSp of the test. The procedures described here can be applied to other tests where meat juice samples are used for testing.     

The development of angiotensin I-converting enzyme inhibitor derived from chicken bone protein

In order to produce angiotensin I‐converting enzyme (ACE) inhibitor for application in functional food, chicken bones were gathered from a meat processing factory and then hydrolyzed with Alcalase, pepsin and trypsin for 12 h. The hydrolysates were lyophilized, stored at ?80°C and tested experimentally every 2 h for pH value, peptide content, degree of hydrolysis (DH), electrophoresis and activity of ACE inhibitor. The hydrolysates of Alcalase had the highest peptide content and DH. The components of more than 66 kDa had disappeared in hydrolysates of Alcalase and trypsin after 2 h of hydrolysis. The hydrolysates of Alcalase were more active in inhibiting ACE, especially when hydrolyzed at 4 and 8 h, and also had low IC50 values of 1.960 and 0.945 mg/mL. According to the results of DH and electrophoresis, the higher activity of ACE inhibitor is assumed to be derived from the low molecular peptides in hydrolysates of Alcalase. Chicken leg bone has a high potential to be utilized to develop ACE inhibitory peptides as a potential ingredient of functional food intended to alleviate hypertension.     

驴皮低温酶解脱毛方法及其对阿胶质量的影响

[目的]开发一种适合阿胶产业化生产的高效驴皮脱毛方法。[方法]低温条件下（4 ℃）,选用1%碱性蛋白酶和1%中性蛋白酶对醒发的干驴皮开展酶解脱毛试验,比较2种酶解方法处理下驴皮的脱毛时间、脱毛率,并对脱毛过程中驴皮的气味进行评价。以1%碱性蛋白酶和1%中性蛋白酶酶解处理驴皮以及机械脱毛鲜驴皮为材料熬制阿胶,比较不同原料熬制阿胶的出胶量、出胶率、外观、气味等;测定以2种蛋白酶酶解处理的驴皮为原料熬制的阿胶质量指标,包括水分、灰分、水不溶物以及4种氨基酸含量,并与《中国药典》2015版一部中提供的相应质量标准进行对比。[结果]低温条件下（4 ℃）,利用1%碱性蛋白酶（pH值=10.5）或1%中性蛋白酶（pH值=7.5）酶解处理驴皮第2天表现出脱毛效果,处理第5天醒发的干驴皮脱毛率可达90%,有臭味产生但非驴皮腐败臭味,而是脱毛过程中产生的正常气味。以1%碱性蛋白酶、1%中性蛋白酶酶解脱毛后的驴皮以及机械脱毛鲜驴皮为原料熬制阿胶,出胶量分别为113.0、109.4、129.3 g,出胶率分别为13.8%、13.1%、13.2%;2种酶解处理驴皮熬制的阿胶色红、气味芳香,与鲜驴皮熬制的阿胶相比色泽更鲜亮。2种酶解处理驴皮熬制阿胶的水分、灰分、水不溶物均符合并优于《中国药典》2015版一部中的质量标准,L-羟脯氨酸、甘氨酸、丙氨酸、L-脯氨酸含量也远高于《中国药典》2015版一部中的质量标准。[结论]低温条件下（4 ℃）,1%碱性蛋白酶和1%中性蛋白酶可对醒发干驴皮进行有效酶解脱毛,且脱毛后驴皮熬制的阿胶质量符合《中国药典》2015版一部标准。